Techniques for the analysis of nucleic acid sequences have found widespread use in basic research, diagnostics, and forensics.
DNA sequencing techniques such as Sanger-Coulson chain terminator sequencing and Maxim and Gilbert sequencing have been used to determine the nucleotide base sequences of large polynucleotides. In many instances, it is of interest to use a simplified technique in order to obtain nucleic acid sequence information about short stretches, e.g., 1 base, of DNA than is typically obtained with sequencing techniques designed to obtain base sequences for comparatively longer regions (e.g., 50-800 bases).
Of particular interest is the use of primer extension techniques that result in the addition of a single nucleotide to the 3' end of polynucleotide probe, such as described in Syvanen et al, Genomics 8, 684-642 (1990). A common embodiment of primer extension analysis adapted for the identification of simple polynucleotides is often referred to as "mini-sequencing." Single base polynucleotide extension techniques such as mini-sequencing are typically used in a solid-phase format in which a binding moiety on the primer or the chain terminators becomes immobilized on a solid phase so as to provide for the detection of the incorporated terminator. The inventors have discovered that differences in the incorporation rate of different labeled polynucleotide chain terminators, can substantially reduce the accuracy and reliability of chain terminator extension. Primer extension techniques such as mini-sequencing employing a fluorescence detecting electrophoresis apparatus, e.g., an automated DNA sequencer, are particularly sensitive to differences in the incorporation rate, e.g., the inability to distinguish heterozygotes from nucleotide misincorporation. The invention described herein greatly reduces the problems associated with differential incorporation of labeled terminators.